Organization and Dynamics of Cholesterol Crystalline Domains using EPR Spin-Labeling

Abstract

EPR spin-labeling methods were used to study the organization and dynamics of cholesterol molecules in membranes formed from Chol/PL (cholesterol/phospholipid) mixtures. Membranes made from phospholipids with different cholesterol solubility thresholds (CST) were investigated. It was confirmed using the EPR discrimination methods that cholesterol crystalline domains (CCDs) were present in all of the membrane suspensions when the mixing ratio exceeded the CST. The behavior of phospholipids was monitored with phospholipid analogue spin labels (n-PCs), and cholesterol with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio exceeding significantly the CST. EPR spectra for n-PC indicated that phospholipids exist in these suspensions in the lipid-bilayer-like structures. Spectral characteristics of n-PCs (spin labels located outside the CCD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CCD forms an integral part of the phospholipid bilayer when formed from Chol/PL mixture up to a mixing ratio of 2 to 3. EPR spectra for CSL and ASL in the CCD and the PCD were very similar indicating that in both domains cholesterol exists in the lipid-bilayer-like structures. In the CCD cholesterol molecules are more ordered and sense more rigid environment than in the PCD. This difference is small and can be compared to that induced in the PCD by the ∼10°C decrease of temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CCD what should enhance their active interaction with the PCD. It is suggested that the EPR spin-labeling approach can discriminate the fraction of cholesterol that forms the CCD within the phospholipid bilayer from the fraction that forms the cholesterol structures outside the bilayer.